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1.  Single-rooted primary first mandibular molar 
BMJ Case Reports  2014;2014:bcr2014206347.
Morphological variations like single-rooted molar in primary dentition are scarce. Understanding the root canal anatomy and variations is necessary for successful root canal therapy. The purpose of the present article is to report successful endodontic treatment of primary left mandibular first molar with an abnormal morphology of a single root. This case report highlights the importance of knowledge and its applications in the management of anomalous anatomic variants which play a crucial role in the success of endodontic treatment.
doi:10.1136/bcr-2014-206347
PMCID: PMC4154040  PMID: 25150245
2.  Mechanical perturbations trigger endothelial nitric oxide synthase activity in human red blood cells 
Scientific Reports  2016;6:26935.
Nitric oxide (NO), a vascular signaling molecule, is primarily produced by endothelial NO synthase. Recently, a functional endothelial NO synthase (eNOS) was described in red blood cells (RBC). The RBC-eNOS contributes to the intravascular NO pool and regulates physiological functions. However the regulatory mechanisms and clinical implications of RBC-eNOS are unknown. The present study investigated regulation and functions of RBC-eNOS under mechanical stimulation. This study shows that mechanical stimuli perturb RBC membrane, which triggers a signaling cascade to activate the eNOS. Extracellular NO level, estimated by the 4-Amino-5-Methylamino-2′, 7′-Difluorofluorescein Diacetate probe, was significantly increased under mechanical stimuli. Immunostaining and western blot studies confirmed that the mechanical stimuli phosphorylate the serine 1177 moiety of RBC-eNOS, and activates the enzyme. The NO produced by activation of RBC-eNOS in vortexed RBCs promoted important endothelial functions such as migration and vascular sprouting. We also show that mechanical perturbation facilitates nitrosylation of RBC proteins via eNOS activation. The results of the study confirm that mechanical perturbations sensitize RBC-eNOS to produce NO, which ultimately defines physiological boundaries of RBC structure and functions. Therefore, we propose that mild physical perturbations before, after, or during storage can improve viability of RBCs in blood banks.
doi:10.1038/srep26935
PMCID: PMC4921846  PMID: 27345770
3.  Serum Chromium Levels in Type 2 Diabetic Patients and Its Association with Glycaemic Control 
Introduction
Chromium is an essential micronutrient which is required for the normal functioning of insulin and regulation of blood sugar levels. It acts as a vital antioxidant for maintaining insulin homeostasis. In diabetes mellitus, the free radical production is increased and levels of antioxidants like chromium, vanadium, selenium and manganese are reduced. There have been previous studies to suggest that low serum levels of chromium are associated with poorer glycaemic control.
Aim
To study the level of serum chromium in newly diagnosed patients with type 2 diabetes mellitus and its association with glycaemic control.
Materials and Methods
Serum chromium concentration was determined by using inductively coupled Plasma – Optical Emission Spectophotometry in 42 newly diagnosed type 2 diabetes mellitus patients without any pre-existing complications. They were divided into 2 groups – well controlled (HbA1c ≤7.0%) and uncontrolled groups (HbA1c >7.0%).
Results
Mean serum chromium concentration measured in uncontrolled type 2 diabetic patients was significantly lower (0.065 ± 0.03 mcg/L vs 0.103 ± 0.04 mcg/L, p< 0.05). There was a statistically significant inverse linear correlation of the HbA1c values and the serum chromium concentration (r= -0.6514, p < 0.0001). There was also a decrease in chromium levels across both the groups with advancing age and the decrease being significant beyond 40 years of age (p<0.05).
Conclusion
The results of our study describes the relationship between serum chromium levels and control of type 2 diabetes mellitus. Significant reduction in chromium levels are probable indicators of metabolic response to oxidative stress in patients with type 2 diabetes mellitus. Further large scale studies relating serum chromium and type 2 diabetes mellitus may help to understand more about the exact relationship.
doi:10.7860/JCDR/2015/16062.6753
PMCID: PMC4668447  PMID: 26676175
Hyperglycemia; Microvascular complications; Retinopathy
4.  Co-Incidence or Co-Existence? Acute Lymphoblastic Leukaemia in HbE-alpha Thalassaemia: A Case Report with Review of Literature 
Haemoglobin E (HbE) is a Haemoglobin variant that commonly occurs in many places in Asia. As β thalassaemia and α thalassaemia also occur in the same regions, the co-inheritance of these conditions leads to various phenotypic forms. HbE α thalassaemia is less common and of a milder phenotype than HbE β thalassaemia. Though malignancies are one of the complications in thalassaemia, occurrence of leukaemia is a rare event. Here we present a case of a two-year-old male child co-presenting with pre B acute lymphoblastic leukaemia (ALL) with MLL rearrangement and HbE alpha thalassaemia. The child is on remission 12 months post-therapy with standard ALL high risk protocol with no minimal residual disease (MRD). Haematological and oncological conditions coexisting at presentation is a challenge to therapy. This case is described for its rarity. Informed consent has been obtained from the parents.
doi:10.7860/JCDR/2015/14711.6796
PMCID: PMC4668511  PMID: 26672845
Haemoglobin variants; Paediatric leukaemia; Thalassaemic syndrome
5.  Biological Reference Interval for Hematological Profile of Umbilical Cord Blood: A Study Conducted at A Tertiary Care Centre in South India 
Introduction
Umbilical cord blood (UCB) a source of hematopoietic stem cells, is also an acceptable sample to assess neonatal sepsis. Though reports are available for stem cell counts very minimal literature is available regarding hematologic parameters, which may vary on ethnicity.
Aim
To establish biological reference interval for hematological parameters of umbilical cord blood to guide neonatologists, hematopoietic stem cell transplant specialists and future analysis.
Materials and Methods
Prospective longitudinal study was done from January 2014 to April 2014 after ethics committee approval. UCB from 120 full term new borns of normal birth weight born out of uneventful pregnancy to mothers aged between 21 to 45 years with hemoglobin above 10g/dL were processed in Beckman Coulter LH780 analyzer for complete blood count and counter checked by peripheral smear. Results tabulated in Microsoft excel are analyzed using IBM SPSS statistics 16 software.
Results
Male to female ratio is 1:1.05. There is no difference in the values between males and females. When compared with few studies available, though many values are comparable a few values are not comparable.
Conclusion
This study can be a useful guide to neonatologists, hematopoietic stem cells transplant hematologists and future analysis.
doi:10.7860/JCDR/2015/14713.6675
PMCID: PMC4625303  PMID: 26557584
Hematology parameters; Reference range; Stem cell transplant
6.  Nimesulide Induced Histopathological Changes in the Vas Deferens of Mice 
Aim: Nimesulide, a preferential COX-2 inhibitor has 20 times more selectivity towards COX-2 than that of COX-1. COX-2 selective inhibitors cause frequent nephrotoxicity and hepatotoxicity following their usage. This proposes a physiological role of COX-2 in kidney and liver. Not much attention has been focused on the role of COX-2 with respect to reproduction especially in male reproduction, and the available information is scanty.
Aims and Objectives: The present study aims to investigate the adverse effects of nimesulide in the vas deferens thereby indirectly assess the role of COX-2 in male reproductive tract.
Material and Methods: Nimesulide was administered orally and the animals were maintained for different time periods prior to sacrifice.
Results: The vas deferens of nimesulide treated mice showed extensive histopathological changes such as vacoulation and exfoliation of cells in the epithelial layer.
Conclusion: Nimesulide administration leads to cytotoxic effects suggestive of apoptosis in the vas deferens of mice.
doi:10.7860/JCDR/2013/6693.3446
PMCID: PMC3843396  PMID: 24298452
Nimesulide; Vas deferens; COX-2; Mice
7.  Marfan Syndrome: A Case Report 
Case Reports in Dentistry  2012;2012:595343.
Marfan syndrome is an autosomal dominant systemic disorder of the connective tissue. Children affected by the Marfan syndrome carry a mutation in one of their two copies of the gene that encodes the connective tissue protein fibrillin-1. Marfan syndrome affects most organs and tissues, especially the skeleton, lungs, eyes, heart, and the large blood vessel that distributes blood from the heart to the rest of the body. A case report of Marfan syndrome has been reported with oral features. The dental problems of the child were treated under general anesthesia and a one-month review showed intact stainless steel crowns' restorations and no signs of secondary caries.
doi:10.1155/2012/595343
PMCID: PMC3529425  PMID: 23304566
8.  EpideMiology and control measures of outBreaks due to Antibiotic-Resistant orGanisms in EurOpe (EMBARGO): a systematic review protocol 
BMJ Open  2017;7(1):e013634.
Introduction
Improving our understanding of outbreaks due to antibiotic-resistant bacteria (ARB) and their control is critical in the current public health scenario. The threat of outbreaks due to ARB requires multifaceted efforts. However, a global overview of epidemiological characteristics of outbreaks due to ARB and effective infection control measures is missing. In this paper, we describe the protocol of a systematic review aimed at mapping and characterising the epidemiological aspects of outbreaks due to ARB and infection control measures in European countries.
Methods and analysis
The databases MEDLINE, Web of Knowledge and Cochrane library will be searched using a 3-step search strategy. Selection of articles for inclusion will be performed by 2 reviewers using predefined eligibility criteria. All study designs will be included if they report an outbreak and define the microbiological methods used for microorganism identification. The target bacteria will be methicillin-resistant and vancomycin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, ceftazidime-resistant and carbapenem-resistant Acinetobacter baumannii, ceftazidime-resistant and carbapenem-resistant Pseudomonas aeruginosa, ciprofloxacin-resistant Escherichia coli, extended-spectrum β-lactamase-producing E. coli and Klebsiella pneumoniae, carbapenem-resistant and carbapenamase-producing Enterobacteriaceae. Data will be extracted using a tailored pilot tested form and the quality of reporting will be assessed using the ORION (Outbreak Reports and Intervention Studies Of Nosocomial infections) tool. Data will be synthesised and reported by the type of ARB, setting and country. Infection control measures and bundles of measures will be described. The effectiveness will be reported as defined by the authors. Regression analysis will be used to define independent factors associated with outbreaks' control. Heterogeneity between studies will be assessed by forest plots and I² statistics.
Ethics and dissemination
Ethical approval is not applicable for this study. Findings will be disseminated through journal publication and conference presentations and talks.
doi:10.1136/bmjopen-2016-013634
PMCID: PMC5223682  PMID: 28057656
outbreak; antimicrobial resistance; EPIDEMIOLOGY
9.  Histone and Non-Histone Targets of Dietary Deacetylase Inhibitors 
Acetylation is an important, reversible post-translational modification affecting histone and non-histone proteins with critical roles in gene transcription, DNA replication, DNA repair, and cell cycle progression. Key regulatory enzymes include histone deacetylase (HDACs) and histone acetyltransferases (HATs). Overexpressed HDACs have been identified in many human cancers, resulting in repressed chromatin states that interfere with vital tumor suppressor functions. Inhibition of HDAC activity has been pursued as a mechanism for re-activating repressed genes in cancers, with some HDAC inhibitors showing promise in the clinical setting. Dietary compounds and their metabolites also have been shown to modulate HDAC activity or expression. Out of this body of research, attention increasingly has shifted towards non-histone targets of HDACs and HATs, such as transcriptions factors, hormone receptors, DNA repair proteins, and cytoskeletal components. These aspects are covered in present review, along with the possible clinic significance. Where such data are available, examples are cited from the literature of studies with short chain fatty acids, polyphenols, isoflavones, indoles, organosulfur compounds, organoselenium compounds, sesquiterpene lactones, isoflavones, and various miscellaneous agents. By virtue of their effects on both histone and non-histone proteins, dietary chemopreventive agents modulate the cellular acetylome in ways that are only now becoming apparent. A better understanding of the molecular mechanisms will likely enhance the potential to more effectively combat diseases harboring altered epigenetic landscapes and dysregulated protein signaling.
Dietary chemopreventive agents modulate the cellular acetylome by affecting both histone and non-histone proteins, which will likely enhance their potential to more effectively combat diseases harboring altered epigenetic landscapes.
Graphical abstract
PMCID: PMC5087604  PMID: 26303421
Acetylation; diet; epigenetics; phytochemicals; HDAC; non-histone
10.  Use of Lung Ultrasound For Diagnosing Acute Heart Failure in Emergency Department of Southern India 
Introduction
Diagnosing heart failure is often a challenge for the healthcare providers due to it’s non-specific and usually subtle physical presentations. The outcomes for treatment are strongly related to the stage of the disease. Considering the importance of early and accurate diagnosis, it is important to have an easy, inexpensive, non-invasive, reliable and reproducible method for diagnosis of heart failure. Recent advancement in radiology and cardiology are supporting the emerging technique of lung ultrasound through B-line evaluation for identifying extravascular lung water.
Aim
To establish lung ultrasound as an easy, inexpensive, non-invasive, reliable and reproducible method for diagnosing Acute Decompensated Heart Failure (ADHF) in emergency department.
Materials and Methods
The study was a cross-sectional, prospective, observational, diagnostic validation study of lung ultrasound for diagnosis of acute heart failure in an emergency department and was performed at Amrita Institute of Medical Science, Kochi, Kerala, India.
A total of 42 patients presenting with symptoms suggestive of acute decompensated heart failure were evaluated by plasma B-type Natriuretic Peptide (BNP), Echocardiography (ECHO) and X-ray. Lung ultrasound was done to look for the presence of B-lines.
Statistical Analysis
Sensitivity, specificity and predictive value of diagnostic modalities were calculated using Mc Nemar’s Chi-square test for the presence and absence of heart failure.
Results
Lung ultrasound showed a sensitivity of 91.9% and a specificity of 100% in diagnosing acute heart failure comparable to plasma BNP which had a sensitivity of 100% and a specificity of 60%. It was also superior to other methods of diagnosing ADHF namely X-ray and ECHO and showed a good association.
Conclusion
Lung ultrasound and its use to detect ultrasonographic B-lines is an early, sensitive and an equally accurate predictor of ADHF in the emergency setting as compared to BNP.
doi:10.7860/JCDR/2016/20661.8814
PMCID: PMC5198425  PMID: 28050472
Accurate predictor; B-lines; B-type natriuretic peptide; Radiology
11.  Modified Technique for Making Auto-polymerized Polymethylmethacrylate Resin Custom Tray 
Custom made tray for dental impression is designed to provide a uniform space for the impression material and thereby improve the accuracy of the resultant working cast. Auto-polymerized acrylic resins have been the most commonly used material for the fabrication of these trays. The custom tray produces more accurate and reliable results for inter-abutment distance at the occlusal and gingival level than stock trays. This article describes a modified technique for fabrication of auto-polymerized Polymethylmethacrylate (PMMA) resin trays.
doi:10.7860/JCDR/2016/21471.8936
PMCID: PMC5198478  PMID: 28050525
Acrylic resin; Dental impression materials; Dental impression techniques
12.  Modified Deacetylcephalosporin C Synthase for the Biotransformation of Semisynthetic Cephalosporins 
Applied and Environmental Microbiology  2016;82(13):3711-3720.
ABSTRACT
Deacetylcephalosporin C synthase (DACS), a 2-oxoglutarate-dependent oxygenase synthesized by Streptomyces clavuligerus, transforms an inert methyl group of deacetoxycephalosporin C (DAOC) into an active hydroxyl group of deacetylcephalosporin C (DAC) during the biosynthesis of cephalosporin. It is a step which is chemically difficult to accomplish, but its development by use of an enzymatic method with DACS can facilitate a cost-effective technology for the manufacture of semisynthetic cephalosporin intermediates such as 7-amino-cephalosporanic acid (7ACA) and hydroxymethyl-7-amino-cephalosporanic acid (HACA) from cephalosporin G. As the native enzyme showed negligible activity toward cephalosporin G, an unnatural and less expensive substrate analogue, directed-evolution strategies such as random, semirational, rational, and computational methods were used for systematic engineering of DACS for improved activity. In comparison to the native enzyme, several variants with improved catalytic efficiency were found. The enzyme was stable for several days and is expressed in soluble form at high levels with significantly higher kcat/Km values. The efficacy and industrial scalability of one of the selected variants, CefFGOS, were demonstrated in a process showing complete bioconversion of 18 g/liter of cephalosporin G into deacetylcephalosporin G (DAG) in about 80 min and showed reproducible results at higher substrate concentrations as well. DAG could be converted completely into HACA in about 30 min by a subsequent reaction, thus facilitating scalability toward commercialization. The experimental findings with several mutants were also used to rationalize the functional conformation deduced from homology modeling, and this led to the disclosure of critical regions involved in the catalysis of DACS.
IMPORTANCE 7ACA and HACA serve as core intermediates for the manufacture of several semisynthetic cephalosporins. As they are expensive, a cost-effective enzyme technology for the manufacture of these intermediates is required. Deacetylcephalosporin C synthase (DACS) was identified as a candidate enzyme for the development of technology from cephalosporin G in this study. Directed-evolution strategies were employed to enhance the catalytic efficiency of deacetylcephalosporin C synthase. One of the selected mutants of deacetylcephalosporin C synthase could convert high concentrations of cephalosporin G into DAG, which subsequently could be converted into HACA completely. As cephalosporin G is inexpensive and readily available, the technology would lead to a substantial reduction in the cost for these intermediates upon commercialization.
doi:10.1128/AEM.00174-16
PMCID: PMC4907206  PMID: 27084018
14.  High-Dose CD20-Targeted Radioimmunotherapy-Based Autologous Transplantation Improves Outcomes for Persistent Mantle Cell Lymphoma 
British journal of haematology  2015;171(5):788-797.
SUMMARY
Autologous stem cell transplant (ASCT) can improve outcomes for mantle cell lymphoma (MCL) patients, yet relapses are frequent. We hypothesized that high-dose anti-CD20 radioimmunotherapy (RIT)-based conditioning could improve results in this setting. We thus assessed 162 consecutive patients with MCL at our centre undergoing ASCT following high-dose RIT-based (n = 61) or standard (n = 101) conditioning. RIT patients were less likely to be in first remission (48% vs 72%; p = 0.002), be in complete remission (CR) (26% vs 61%; p < 0.001) and have chemosensitive disease (84% vs 96%; p = 0.006). RIT-based conditioning was associated with a reduced risk of treatment failure (hazard ratio [HR] 0.40; p = 0.001) and mortality (HR 0.49; p = 0.01) after adjusting for these imbalances. This difference increased as disease status worsened (from CR to partial remission to stable/progressive disease), with respective HRs of 1.14, 0.53 and 0.04 for mortality, and 0.66, 0.36 and 0.14 for treatment failure. RIT-based conditioning appears to improve outcome following ASCT for MCL patients unable to achieve CR after controlling for imbalances in important risk factors. These data support the further study of RIT and radiation-based strategies in a risk-adapted approach to ASCT for persistent MCL.
doi:10.1111/bjh.13773
PMCID: PMC4715476  PMID: 26455717
non-Hodgkin lymphoma; stem cell transplantation; antibody therapy; radiotherapy
15.  A Case of Interosseous Membrane Calcification 
doi:10.7860/JCDR/2016/22342.8619
PMCID: PMC5121724  PMID: 27891386
Fluorosis; Metastatic calcification; Renal Osteodystrophy
16.  CD137 and CD137L signals are main drivers of type 1, cell-mediated immune responses 
Oncoimmunology  2015;5(4):e1113367.
ABSTRACT
CD137 is expressed on activated T cells and NK cells, among others, and is a potent co-stimulator of antitumor immune responses. CD137 ligand (CD137L) is expressed by antigen presenting cells (APC), and CD137L reverse signaling into APC enhances their activity. CD137–CD137L interactions as main driver of type 1, cell-mediated immune responses explains the puzzling observation that CD137 agonists which enhance antitumor immune responses also ameliorate autoimmune diseases. Upon co-stimulation by CD137, Th1 CD4+ T cells together with Tc1 CD8+ T cells and NK cells inhibit other T cell subsets, thereby promoting antitumor responses and mitigating non-type 1 auto-immune diseases.
doi:10.1080/2162402X.2015.1113367
PMCID: PMC4839363  PMID: 27141396
CD137; T cell polarization; Tc1; Th1
17.  A novel targeted/untargeted GC-Orbitrap metabolomics methodology applied to Candida albicans and Staphylococcus aureus biofilms 
Metabolomics  2016;12(12):189.
Introduction
Combined infections from Candida albicans and Staphylococcus aureus are a leading cause of death in the developed world. Evidence suggests that Candida enhances the virulence of Staphylococcus—hyphae penetrate through tissue barriers, while S. aureus tightly associates with the hyphae to obtain entry to the host organism. Indeed, in a biofilm state, C. albicans enhances the antimicrobial resistance characteristics of S. aureus. The association of these microorganisms is also associated with significantly increased morbidity and mortality. Due to this tight association we hypothesised that metabolic effects were also in evidence.
Objectives
To explore the interaction, we used a novel GC-Orbitrap-based mass spectrometer, the Q Exactive GC, which combines the high peak capacity and chromatographic resolution of gas chromatography with the sub-ppm mass accuracy of an Orbitrap system. This allows the capability to leverage the widely available electron ionisation libraries for untargeted applications, along with expanding accurate mass libraries and targeted matches based around authentic standards.
Methods
Optimised C. albicans and S. aureus mono- and co-cultured biofilms were analysed using the new instrument in addition to the fresh and spent bacterial growth media.
Results
The targeted analysis experiment was based around 36 sugars and sugar phosphates, 22 amino acids and five organic acids. Untargeted analysis resulted in the detection of 465 features from fresh and spent medium and 405 from biofilm samples. Three significantly changing compounds that matched to high scoring library fragment patterns were chosen for validation.
Conclusion
Evaluation of the results demonstrates that the Q Exactive GC is suitable for metabolomics analysis using a targeted/untargeted methodology. Many of the results were as expected: e.g. rapid consumption of glucose and fructose from the medium regardless of the cell type. Modulation of sugar-phosphate levels also suggest that the pentose phosphate pathway could be enhanced in the cells from co-cultured biofilms. Untargeted metabolomics results suggested significant production of cell-wall biosynthesis components and the consumption of non-proteinaceous amino-acids.
Electronic supplementary material
The online version of this article (doi:10.1007/s11306-016-1134-2) contains supplementary material, which is available to authorized users.
doi:10.1007/s11306-016-1134-2
PMCID: PMC5097782  PMID: 28003796
Metabolomics; Candida; Staphylococcus; Biofilm
18.  SYNTAX Score in Patients with High Computed Tomography Coronary Calcium Score 
Objectives:
To study the conventional coronary angiogram ( CA) findings in patients with high coronary calcium on multidetector computed tomogram.
Materials and Methods:
Fifty patients with coronary calcium high enough in its extent and location to interfere with the interpretation of a contrast-filled coronary artery for a significant lesion were studied with conventional CA. Framingham risk score (FRS), computed tomography (CT) coronary calcium score (CCS), and SYNTAX score (SS) from the CA were calculated by separate investigators who were blinded to other scores. Effectively, 250 coronary arteries (left main, left anterior descending, left circumflex, and right coronary artery and posterior descending artery in each subject) with calcium scores were studied for lesions on CA.
Results:
Thirty-five subjects had high FRS, 10 had intermediate FRS, and 5 had low FRS. Eight subjects of 25 (32%) with CCS between 350 and 1000 had no significant coronary artery disease (CAD). Overall, the CCS and the SS had a strong agreement with each other (r = 0.68, P < 0.01) that persisted in those with very high scores >1000 (r = 0.55, P < 0.01, n = 30), but only a nonsignificant weak correlation with scores between 350 and 1000 (r = 0.1, P = 0.62, n = 20). Individual vessel calcium scores correlated strongly for the presence of any lesion (r = 0.52, P < 0.01) in the same artery but only weakly for a significant lesion (r = 0.29, P = 0.05).
Conclusion:
High CT CCS in this cohort of intermediate to high (Framingham score) risk patients correlated strongly with the subject's global burden of the CAD as derived by the SS, more so for subjects with very high scores. Similarly, CCS correlated strongly with the presence of any lesion but only weakly for a significant stenosis; also, about one-third of patients with CCS between 350 and 1000 may not have significant disease on conventional CA.
doi:10.4103/2156-7514.193423
PMCID: PMC5156969  PMID: 28028450
Calcium score; coronary computed tomography angiogram; Framingham risk score; risk stratification; SYNTAX score
19.  The Endothelial Prolyl-4-Hydroxylase Domain 2/Hypoxia-Inducible Factor 2 Axis Regulates Pulmonary Artery Pressure in Mice 
Molecular and Cellular Biology  2016;36(10):1584-1594.
Hypoxia-inducible factors 1 and 2 (HIF-1 and -2) control oxygen supply to tissues by regulating erythropoiesis, angiogenesis and vascular homeostasis. HIFs are regulated in response to oxygen availability by prolyl-4-hydroxylase domain (PHD) proteins, with PHD2 being the main oxygen sensor that controls HIF activity under normoxia. In this study, we used a genetic approach to investigate the endothelial PHD2/HIF axis in the regulation of vascular function. We found that inactivation of Phd2 in endothelial cells specifically resulted in severe pulmonary hypertension (∼118% increase in right ventricular systolic pressure) but not polycythemia and was associated with abnormal muscularization of peripheral pulmonary arteries and right ventricular hypertrophy. Concurrent inactivation of either Hif1a or Hif2a in endothelial cell-specific Phd2 mutants demonstrated that the development of pulmonary hypertension was dependent on HIF-2α but not HIF-1α. Furthermore, endothelial HIF-2α was required for the development of increased pulmonary artery pressures in a model of pulmonary hypertension induced by chronic hypoxia. We propose that these HIF-2-dependent effects are partially due to increased expression of vasoconstrictor molecule endothelin 1 and a concomitant decrease in vasodilatory apelin receptor signaling. Taken together, our data identify endothelial HIF-2 as a key transcription factor in the pathogenesis of pulmonary hypertension.
doi:10.1128/MCB.01055-15
PMCID: PMC4859687  PMID: 26976644
20.  The H-ARS Dose Response Relationship (DRR): Validation and Variables 
Health physics  2015;109(5):391-398.
Manipulations of lethally-irradiated animals, such as for administration of pharmaceuticals, blood sampling, or other laboratory procedures, have the potential to induce stress effects that may negatively affect morbidity and mortality. To investigate this in a murine model of the hematopoietic acute radiation syndrome, 20 individual survival efficacy studies were grouped based on the severity of the administration (Admn) schedules of their medical countermeasure (MCM) into Admn 1 (no injections), Admn 2 (one to three injections), or Admn 3 (29 injections or six to nine oral gavages). Radiation doses ranged from LD30/30 to LD95/30. Thirty-day survival of vehicle controls in each group was used to construct radiation dose lethality response relationship (DRR) probit plots, which were compared statistically to the original DRR from which all LDXX/30 for the studies were obtained. The slope of the Admn 3 probit was found to be significantly steeper (5.190) than that of the original DRR (2.842) or Admn 2 (2.009), which were not significantly different. The LD50/30 for Admn 3 (8.43 Gy) was less than that of the original DRR (8.53 Gy, p<0.050), whereas the LD50/30 of other groups were similar. Kaplan-Meier survival curves showed significantly worse survival of Admn 3 mice compared to the three other groups (p=0.007). Taken together, these results show that stressful administration schedules of MCM can negatively impact survival, and that dosing regimens should be considered when constructing DRR to use in survival studies.
doi:10.1097/HP.0000000000000354
PMCID: PMC4593318  PMID: 26425900
Health effects; mice; radiation dose; radiation damage
21.  DELAYED EFFECTS OF ACUTE RADIATION EXPOSURE IN A MURINE MODEL OF THE H-ARS: MULTIPLE-ORGAN INJURY CONSEQUENT TO <10 GY TOTAL BODY IRRADIATION 
Health physics  2015;109(5):511-521.
The threat of radiation exposure from warfare or radiation accidents raises the need for appropriate animal models to study the acute and chronic effects of high dose rate radiation exposure. The goal of this study was to assess the late development of fibrosis in multiple organs (kidney, heart, and lung) in survivors of the C57BL/6 mouse model of the hematopoietic-acute radiation syndrome (H-ARS). Separate groups of mice for histological and functional studies were exposed to a single uniform total body dose between 8.53 and 8.72 Gy of gamma radiation from a 137Cs radiation source and studied 1–21 months later. Blood urea nitrogen levels were elevated significantly in the irradiated mice at 9 and 21 mo (from ~22 to 34 ± 3.8 and 69±6.0 mg/dl, p<0.01 vs non-irradiated controls) and correlated with glomerosclerosis (29±1.8% vs 64±9.7% of total glomeruli, p<0.01 vs non-irradiated controls). Glomerular tubularization and hypertrophy and tubular atrophy were also observed at 21 mo post-total body irradiation (TBI). An increase in interstitial, perivascular, pericardial and peri-bronchial fibrosis/collagen deposition was observed from ~9–21 mo post-TBI in kidney, heart and lung of irradiated mice relative to age-matched controls. Echocardiography suggested decreased ventricular volumes with a compensatory increase in left ventricular ejection fraction. The results indicate that significant delayed effects of acute radiation exposure occur in kidney, heart, and lung in survivors of the murine H-ARS TBI model which mirrors pathology detected in larger species and humans at higher radiation doses focused on specific organs.
doi:10.1097/HP.0000000000000357
PMCID: PMC4593322  PMID: 26425910
Health effects; mice; radiation dose; radiation damage
22.  Variations in the Origin and Course of Right Hepatic Artery and its Surgical Significance 
Introduction
Variations in the Right Hepatic Artery (RHA) are exceedingly common and these variations are of great importance to surgeons and interventional radiologists. A thorough knowledge of the right hepatic arterial anatomy is mandatory while performing hepatic surgery and hepatic arteriography.
Aim
To find out the variations in the origin and course of the RHA and its surgical significance.
Materials and Methods
This study was conducted in 60 embalmed adult cadavers of both sexes of age group between 50 to 80 years. Abdomen was opened by a linear midline incision extending from the xiphoid process to the pubic symphysis. The origin of RHA from the proper hepatic artery was traced. The presence of aberrant right hepatic arteries -replaced and accessory were noted. The source of origin of accessory or replaced RHA arising from the superior mesenteric artery (SMA), right renal artery and inferior mesenteric artery was traced. The course of normal and aberrant RHA from its origin to the entrance into the right lobe of the liver was followed and structures related to it were noted.
Results
The RHA mostly originated from the main trunk of the proper hepatic artery in 52 specimens (86.6%). Presence of aberrant RHA was found in 8 specimens (13.3%). Among 8 specimens of aberrant RHA, replaced RHA arising from SMA was found in 5 specimens (8.3%) and accessory RHA arising from SMA was observed in 3 specimens (5%). In 86.6% of specimens, the RHA coursed dorsal to the duct system to enter the Calot’s triangle. In 8.3% of specimens, the RHA coursed ventral to duct system to reach the Calot’s triangle. The caterpillar like loop of the RHA was found in two specimens (3%). The replaced and accessory RHA (13.3%) arising from the SMA had a course dorsal to duct system to reach the Calot’s triangle.
Conclusion
Right hepatic artery is subject to anatomical variation in its origin and course. Our study identified the variations in the origin and course of right hepatic artery. Adequate knowledge of these variations would be of incredible help to the hepatobiliary surgeon and interventional radiologist.
doi:10.7860/JCDR/2016/22126.8428
PMCID: PMC5071913  PMID: 27790413
Aberrant; Accessory; Anatomy; Cadaver; Human; Liver; Replaced
23.  Complex Breakpoints and Template Switching Associated with Non-canonical Termination of Homologous Recombination in Mammalian Cells 
PLoS Genetics  2016;12(11):e1006410.
A proportion of homologous recombination (HR) events in mammalian cells resolve by “long tract” gene conversion, reflecting copying of several kilobases from the donor sister chromatid prior to termination. Cells lacking the major hereditary breast/ovarian cancer predisposition genes, BRCA1 or BRCA2, or certain other HR-defective cells, reveal a bias in favor of long tract gene conversion, suggesting that this aberrant HR outcome might be connected with genomic instability. If termination of gene conversion occurs in regions lacking homology with the second end of the break, the normal mechanism of HR termination by annealing (i.e., homologous pairing) is not available and termination must occur by as yet poorly defined non-canonical mechanisms. Here we use a previously described HR reporter to analyze mechanisms of non-canonical termination of long tract gene conversion in mammalian cells. We find that non-canonical HR termination can occur in the absence of the classical non-homologous end joining gene XRCC4. We observe obligatory use of microhomology (MH)-mediated end joining and/or nucleotide addition during rejoining with the second end of the break. Notably, non-canonical HR termination is associated with complex breakpoints. We identify roles for homology-mediated template switching and, potentially, MH-mediated template switching/microhomology-mediated break-induced replication, in the formation of complex breakpoints at sites of non-canonical HR termination. This work identifies non-canonical HR termination as a potential contributor to genomic instability and to the formation of complex breakpoints in cancer.
Author Summary
Complex breakpoints are a recognized feature of cancer genome rearrangements, but the mechanisms that lead to their formation are undefined. Although homologous recombination (HR) is considered a potentially error-free pathway, cells lacking critical HR genes, such as the major hereditary breast/ovarian cancer predisposition genes, BRCA1 or BRCA2, frequently engage error-prone homologous recombination mechanisms in which HR termination does not occur in a timely fashion. We show here that aberrant termination of HR in mammalian cells involves the use of error-prone alternative end joining mechanisms and can lead to the formation of complex breakpoints by means of template switching mechanisms. This suggests that defective termination of homologous recombination underlies some of the complex breakpoints observed in cancer cells.
doi:10.1371/journal.pgen.1006410
PMCID: PMC5104497  PMID: 27832076
24.  Effect of High-Carbohydrate Diet on Plasma Metabolome in Mice with Mitochondrial Respiratory Chain Complex III Deficiency 
Mitochondrial disorders cause energy failure and metabolic derangements. Metabolome profiling in patients and animal models may identify affected metabolic pathways and reveal new biomarkers of disease progression. Using liver metabolomics we have shown a starvation-like condition in a knock-in (Bcs1lc.232A>G) mouse model of GRACILE syndrome, a neonatal lethal respiratory chain complex III dysfunction with hepatopathy. Here, we hypothesized that a high-carbohydrate diet (HCD, 60% dextrose) will alleviate the hypoglycemia and promote survival of the sick mice. However, when fed HCD the homozygotes had shorter survival (mean ± SD, 29 ± 2.5 days, n = 21) than those on standard diet (33 ± 3.8 days, n = 30), and no improvement in hypoglycemia or liver glycogen depletion. We investigated the plasma metabolome of the HCD- and control diet-fed mice and found that several amino acids and urea cycle intermediates were increased, and arginine, carnitines, succinate, and purine catabolites decreased in the homozygotes. Despite reduced survival the increase in aromatic amino acids, an indicator of liver mitochondrial dysfunction, was normalized on HCD. Quantitative enrichment analysis revealed that glycine, serine and threonine metabolism, phenylalanine and tyrosine metabolism, and urea cycle were also partly normalized on HCD. This dietary intervention revealed an unexpected adverse effect of high-glucose diet in complex III deficiency, and suggests that plasma metabolomics is a valuable tool in evaluation of therapies in mitochondrial disorders.
doi:10.3390/ijms17111824
PMCID: PMC5133825  PMID: 27809283
mitochondrial disorder; BCS1L; mouse model; metabolite; dextrose diet; nutrition; GRACILE syndrome
25.  Lipid-based surface engineering of PLGA nanoparticles for drug and gene delivery applications 
Biomaterials Research  2016;20:34.
The use of poly(lactic-co-glycolic acid) (PLGA)-based nanocarriers presents several major challenges, including their synthetic hydrophobic surface, low transfection efficiency, short circulation half-life, and nonspecific tissue distribution. Numerous engineering strategies have been employed to overcome these problems, with lipid-based surface functionalization of PLGA nanoparticles (NPs) showing promising results in the development of PLGA-based clinical nanomedicines. Surface engineering with different lipids enhances the target specificity of the carrier and improves its physicochemical properties as well as NP-cell associations, such as cellular membrane permeability, immune responses, and long circulation half-life in vivo. This review focuses on recent advances in the lipid-based surface engineering of PLGA NPs for drug and gene delivery applications.
doi:10.1186/s40824-016-0081-3
PMCID: PMC5087123  PMID: 27807476
Surface engineering; Lipids; PLGA nanoparticle; Self assembly; Cell membrane derived lipid vesicles; Biomimetic fucntionalization; Controlled drug release; Gene delivery

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